1. Field of the Art
This application generally relates to the construction of PHB-deficient Sphingomonas strains that produce high yields of diutan with improved filterability. In another aspect, this application relates to diutan produced from PHB-deficient Sphingomonas strains that produce high yields of diutan with improved filterability.
2. Description of Related Art
A number of bacteria of the genus Sphingomonas produce polysaccharides called sphingans that have related structures with a generally conserved tetrasaccharide backbone structure and different side chains (ref. no. 1, 6, 7, 8, 10). The sphingans gellan, welan, rhamsan and diutan are produced commercially for use in food, oilfield or personal care applications. The value of sphingan polysaccharides lies in their abilities to modify the rheology of aqueous solutions, i.e., to thicken liquids, suspend solids, stabilize emulsions, or form gels and films.
Sphingans are structurally related to one another, but are not identical. Common members of the genus Sphingomonas and the sphingans they produce include Sphingomonas elodea ATCC 31461, which produces gellan (S-60); Sphingomonas sp. ATCC 31555, which produces welan (S-130); Sphingomonas sp. ATCC 31961, which produces rhamsan (S-194); Sphingomonas sp. ATCC 53159, which produces diutan (S-657); Sphingomonas sp. ATCC 31554, which produces an as yet unnamed polysaccharide (S-88); Sphingomonas sp. ATCC 31853, which produces an as yet unnamed polysaccharide (S-198); Sphingomonas sp. ATCC 21423, which produces an as yet unnamed polysaccharide (S-7); Sphingomonas sp. ATCC 53272, which produces an as yet unnamed polysaccharide (NW-11); Sphingomonas sp. FERM-BP2015 (previously Alcaligenes latus B-16), which produces alcalan (Biopolymer B-16) and the like. A description of the Sphingomonads and the polysaccharides they produce can be found, for example, in U.S. Pat. Nos. 4,377,636; 4,326,053; 4,326,052 and 4,385,123 (for ATCC 31461 and its S-60 polysaccharide); in U.S. Pat. No. 4,342,866 (for ATCC 31555 and S-130); in U.S. Pat. No. 4,401,760 (for ATCC 31961 and S-194); in U.S. Pat. No. 5,175,278 (for ATCC 53159 and S-657); in U.S. Pat. Nos. 4,331,440 and 4,535,153 (for ATCC 31554 and S-88); in U.S. Pat. No. 4,529,797 (for ATCC 31853 and S-198); in U.S. Pat. No. 3,960,832 (for ATCC 21423 and S-7); in U.S. Pat. No. 4,874,044 (for ATCC 53272 and NW-11); in U.S. Pat. No. 5,175,279 (for FERM BP-2015 and B-16), each of which is incorporated by reference herein in its entirety to the extent that they are not inconsistent with the disclosure herein.
One particular sphingan, diutan (also known as heteropolysaccharide S-657), is prepared by fermentation of strain Sphingomonas sp. ATCC 53159 (ref. no. 17). Diutan exhibits unique rheological properties in aqueous solutions including high thermal stability, superior suspension properties, and the ability to generate high viscosity at low concentrations. The diutan polysaccharide imparts significant pseudoplasticity to polar solvents such as water, such that diutan can act as a rheological modifier that is capable of particle suspension, friction reduction, emulsion and foam stabilization, filter cake deposition and filtration control. Consequently, diutan has found industrial utility as a rheological modifier in a variety of contexts, including cementitious systems as disclosed in U.S. Pat. No. 6,110,271, which is incorporated herein by reference in its entirety to the extent that they are not inconsistent with the disclosure herein.
Diutan consists of a repeat unit with a backbone comprised of [→4)-α-L-rhamnose-(1→3)-β-D-glucose-(1→4)-β-D-glucuronic acid-(1→4)-β-D-glucose-(1→] and a two-sugar L-rhamnose side-chain attached to the (1→4) linked glucose residues (ref. no. 2, 7). Two O-acetyl groups are attached per repeat unit to the 2′ and 6′ positions of the (1→3) linked glucose (ref. no. 4).
Progress has been made in elucidating the genetics and biochemistry underlying biosynthesis of diutan and other sphingans. Genes for biosynthesis of sphingans S-88, S-7, and gellan have been identified (ref. no. 5, 12, 13, 15). Genes for several glycosyl transferases of the backbone structure have been analyzed biochemically (ref. no. 11, 14), as have genes gelC and gelE, potentially involved in chain length determination (ref. no. 9). Several of the genes for synthesis of sugar nucleotide precursors have also been elucidated (ref. no. 12). The genetics and biochemistry of polymerization, secretion and control of polysaccharide molecular length are less defined.
A cluster of genes involved in biosynthesis of diutan has been identified that includes genes for glycosyl transferases, genes encoding enzymes for synthesis of a precursor molecule dTDP rhamnose, and genes for secretion of the polysaccharide (ref. no. 3). Plasmids, e.g., pS8 and pX6, containing some of the genes in the aforementioned cluster, were shown to increase the yield of diutan by about 10%, and one plasmid in particular (pS8) was found to significantly improve the rheological properties of diutan from the wild-type strain (ref. no. 18).
Growth conditions typically used for producing diutan and other sphingans also promote production of the internal storage polymer polyhydroxybutyrate (“PHB”), which is generally regarded as an undesirable side-product and is difficult to remove during sphingan preparation. The PHB can form small insoluble particles that interfere with clarity and filterability, limiting the usefulness of the sphingans. For example, the turbidity imparted by PHB particles can limit applicability for household and personal care products in which appearance is critical for consumer acceptance. Moreover, certain oilfield uses require filterability; however, the PHB particles can plug small pores in oil field rock formations, preventing the flow of the sphingan solution and/or the return flow of the crude oil after treating the well. Finally, as PHB synthesis and sphingan synthesis compete for the available carbon source, PHB synthesis can have some adverse effect on sphingan yield.
Accordingly, attempts have been made to eliminate PHB production in sphingan-producing strains. Ref. no. 26 describes a strain of Sphingomonas elodea (a gellan-producing species) that was isolated following chemical mutagenesis. This strain, called LPG-2, has decreased PHB production, but produces gellan of inconsistent quality and yield.
A more targeted approach to eliminating PHB production was undertaken by deletion of a gene required for PHB synthesis, the phaC gene (ref. no. 20). Precise deletion of phaC from a diutan producing strain (ATCC 53159) reproducibly resulted in poor growth and severely reduced diutan productivity (strains NPD3 and NPD6). These strains exhibit increased carbohydrate hydrolysis and accumulation of organic acids, suggesting a critical role for phaC in maintaining normal cellular metabolism. Derivatives with less impaired diutan productivity were subsequently isolated. Two independent derivatives, PDD3 and PDD6, have uncharacterized spontaneous mutation(s) and remain PHB-deficient (ATCC deposit nos. PTA-4865 and PTA-4866, respectively). Though recovery of up to 90% of total diutan yield has been reported (ref. no. 20), this yield was only obtained following a greatly increased culture growth time and has not been consistently reproducible. Under standard growth conditions, diutan productivity and yield by these strains is only approximately half of wild-type levels.